Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

Summary

This protocol describes a noninvasive technique for the sampling of undisturbed mucosal lining fluid from the upper airways. It can be used to perform the quantification of in vivo levels of protein mediators, such as cytokines and chemokines, in subjects of all ages.

Abstract

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the analysis of immune mediators in fluid eluates for the study of the airway topical immune signature, without the need for stimulation procedures (often used by other techniques). The mucosal lining fluid is sampled on a strip of filter paper placed at the anterior part of the inferior turbinate and left for 2 min of absorption. Analytes are eluted from the filter papers, and the extracted protein-based eluates are analyzed by an electrochemiluminescence-based immunoassay, allowing for the high-sensitivity quantification of low- and high-level analytes in the same sample. We measured the in vivo levels of 20 preselected immune mediators related to specific immune signaling pathways in the upper airway mucosa, but the technique is not limited to that specific panel or sampling site. The technique was first implemented in 7-year-old children from the Copenhagen Prospective Studies on Asthma in Childhood₂₀₀₀ (COPSAC₂₀₀₀) cohort with allergic rhinitis. It was thereafter used in the longitudinal COPSAC₂₀₁₀ birth cohort, sampled at 1 month, 2 years, and 6 years of age and at instances of acute respiratory symptoms. We successfully obtained and analyzed samples from 620 (89%) of 700 1-month-old children; a few samples were below the assay detection limit (reported as the median (Inter-Quartile Range (IQR)). The number of samples below the detection limit (i.e. from 0 to the set point for the lower limit of detection) for each mediator was 29 (7.25 - 119.5). This technique enables the quantification of the in vivo airway mucosal immune profile from birth, can be applied longitudinally, and can be applied to studies on the effect of genetics and early-life environmental exposures, pathophysiology, endotyping, and monitoring of respiratory diseases, and development and evaluation of novel therapeutics.

Introduction

The mucosal lining fluid of the nose makes up the liquid part of the upper-airway system. It consists of a complex matrix of mediators derived from the interplay between the epithelium and the immune cells that make up the first line of defense against invading microorganisms. The nasal mucosa is easily accessible, and there is a strong functional and immunological relationship between the nose and the bronchi¹. This compartment is of special interest in relation to airway diseases that are common in childhood, such as asthma and allergic rhinitis, but also to a range of other respiratory disorders more prevalent later in life.

Here, we describe the implementation of a method to sample undisturbed mucosal lining fluid from the nasal cavity using a filter paper-based, noninvasive technique, as well as a subsequent extraction procedure, used to elute protein-based analytes from the filter papers prior to their quantification. This technique can, for example, be used to obtain in vivo immune signatures of both healthy individuals and individuals with various respiratory diseases. Furthermore, it is possible to examine exposures of importance for a specific immune signature and to evaluate if it is a predictor or mediator of later disease development.

Mucosal lining fluid has previously been obtained by nasal lavage², which is often preceded by a nasal challenge test, where an allergen is introduced in high levels to stimulate an inflammatory response^3,4. However, the nasal lavage technique is not feasible in young children and introduces an unknown dilution factor, which confounds the outcomes, as the diluted mediator levels can fall below the detection limit of the assay⁵. Moreover, due to the unknown dilution factor, the measured analyte responses from the nasal challenge tests are not comparable between individuals, thereby limiting the usefulness of the nasal lavage technique in a cohort setting. Finally, allergen challenge is only applicable in sensitized subjects, and other challenges, such as the histamine challenge, are not physiologically relevant, potentially causing a ceiling effect on mediator release. These problems are circumvented in the presented filter paper-based technique for mucosal lining fluid collection, where the individual secretion of fluids and analyte levels are the only factors that influence inter-individual variance.

During the extraction procedure, analytes are eluted from the filter papers after the addition of identical volumes of buffer to all samples. This favors similar ex vivo dilution of all samples. An extraction buffer of albumin-based isotonic salt solution is used for the extraction step; it enables the extraction of protein-based mediators and stabilizes proteins to limit denaturation during the subsequent freezing of eluted proteins prior to quantification. To avoid protein degradation during the extraction phase, a cocktail of protease inhibitors is added to the extraction buffer.

The implementation of techniques that allow for the quantification of undisturbed, in vivo-generated immune mediators at mucosal sites is of the utmost importance. First, the mucosal site makes up the largest immunological organ in the body. Second, the nasal location is the primary site of airborne exposure and is tightly connected to the respiratory immunological compartment of the lungs¹. Third, the possibility of surveying this important organ with a noninvasive technique opens the possibility to provide a plethora of information on the important microbe-immune interaction axis in relation to health and disease in the airways. Fourth, there are many other possible applications of this technique, such as studying local immunological alterations in randomized, controlled trials of drugs and micronutrients.

We initially implemented the technique in the Copenhagen Prospective Studies on Asthma in Childhood₂₀₀₀ (COPSAC₂₀₀₀) cohort, where we determined the immune profile of the mucosal lining fluid in 7-year-old children with allergic rhinitis versus healthy controls¹³. Subsequently, we successfully applied this technique to the longitudinal COPSAC₂₀₁₀ cohort and assessed airway immune profiles at 1 month, 2 years, and 6 years of age and at instances of acute respiratory symptoms. Results from the 1-month-old neonates have demonstrated important associations between the immune signature and early-life environmental exposure^{7,8,9,10,11,12}.

Protocol

The studies were conducted in accordance with the guiding principles of the Declaration of Helsinki. Approvals from the Ethics Committee for Copenhagen (KF 01-289/96 for COPSAC₂₀₀₀ and H-B-2008-093 for COPSAC₂₀₁₀) and the Danish Data Protection Agency were obtained, and written informed consent was obtained from both parents of each subject before enrollment.

1. Experimental Setup

1. Use sheets of filter paper (fibrous hydroxylated-polyester sheets, see the Table of Materials)^6,7.
   1. Cut out strips 3 x 15 mm in size for a 1-month-old child and in an L-shape for older children and adults (5 x 20 x 10 mm (short arm of the L)).
2. Insert one piece of filter paper into each nostril of an infant using forceps. Place the filter paper at the anterior part of the inferior turbinate (approximately 1 cm inside the nostril for neonates and 1.5 cm for all other ages).
   1. Place neonates on a bed and give them sugar water for comfort.
      NOTE: For children of all other ages, the sampling is performed most easily with the subject seated on a chair.
   2. For older children, insert the long arm of the L-shaped filter paper into each nostril.
3. Apply a nose clip around the nostrils to minimize discomfort and to avoid the accidental loss of the filter paper.
4. Remove the filter papers after 2 min of absorption.
5. Place the filter papers in labeled tubes and freeze them immediately at -80 °C.
6. Make a note of any symptoms of airway infection shown by the child on the day of sampling.
7. Record any sneezing, persistent crying, or epistaxis that occurs during the 2 min of sampling.

2. Quantification of Airway Immune-mediators

1. Take up to 10 random samples from the freezer. Record the identification numbers. Keep the samples on ice during the whole working process.
2. After thawing on ice, immerse the filter papers (per subject) from both nostrils in 300 µL of freshly prepared buffer (see the Table of Materials) containing one complete protease inhibitor tablet (see the Table of Materials) per 25 mL of buffer.
   1. Adjust the volume of buffer as per the size of the filter paper.
      1. Use 300 µL of buffer for filter papers 3 x 15 mm in size.
      2. If only one filter paper is available, use half the buffer volume (150 µL).
3. Transfer the samples to a plate shaker (400 rpm) for 5 min. Use a timer.
4. Transfer the moist filter papers and assay buffer into the cup of a cellulose acetate tube filter (0.22 µm pore size) placed within a microcentrifuge tube (see the Table of Materials).
5. Centrifuge at 16,000 x g for 5 min in a cooled centrifuge (at 4 °C) to obtain filtered nasal extract in the tube.
6. Remove the cup and keep the tubes on ice while aliquoting the nasal extract into the wells of low-protein binding storage plates (see the Table of Materials).
7. Store at -80 °C until analysis.
8. Determine the concentrations of cytokines and chemokines in the nasal extracts using a high-sensitivity, electrochemiluminesce-based multiplexed array system (see the Table of Materials).
   NOTE: A human 10-plex T_H1/T_H2 cytokine assay, 9-plex chemokine assay, and single-plex IL-17A, TGF-β1, and TSLP were performed here.
   1. For the assays, incubate the nasal extracts overnight at 4 °C. Conduct the measurements as per the standard manufacturer protocol (see the Table of Materials).
      NOTE: The immunoassays (see the Table of Materials) typically have a high dynamic range for measurement ranging between 1 & 10,000 pg/mL, but for some assays, it can be 100,000 pg/mL. This means that all samples can be run at the same dilution, thus limiting the influence of dissimilar dilutions in healthy and diseased subjects, as is the case in other similar immunoassays. The lower limit of detection for all cytokines was 1 pg/mL or less, and for chemokines, it ranged between 1 & 50 pg/mL. TSLP was not detectable in 98% of samples at 1 month of age.

3. Statistics

1. Log-transform the data prior to analysis to obtain normally distributed residuals of the cytokine and chemokine levels.
2. If the samples are analyzed in more than one batch, correct the cytokine and chemokine data for batch-wise centering on the log-transformed data.
   1. Incorporate the overall mean and anti-log transformation to obtain data in the original measurement units.
      NOTE: For every set of analyses done, consider adjusting variables that affect the level of cytokines and chemokines. This could be maternal influences (e.g., alcohol consumption and smoking during pregnancy), breastfeeding, adverse events during sampling, siblings, pets in the home, etc.

Results

Baseline Characteristics of the Airway Immune Profiles:
Complete data on upper airway mucosal immune mediator levels at 1 month of age was obtained in 620 (89%) of the 700 children enrolled in the COPSAC₂₀₁₀ cohort. Ten neonates were enrolled before the technique was established, and 19 did not attend the 1-month visit. Additionally, 47 samples were excluded because they were extracted and measured in another laboratory used in a pilot study, and 4 samples ...

Discussion

With the technique presented here, we were able to determine the in vivo upper-airway mucosal immune profile in children from as early as 1 month of age, which has not been done previously. We observed that presence of specific airway bacteria and picornaviruses^7,11, as well as other pre and perinatal exposures, were mirrored in the airway immune profile of the neonates. Furthermore, we used these airway immune profile data to study the mechanism of...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fibrous hydroxylatedpolyester sheets	Accuwik Ultra	SPR0730	Filter paper
Milliplex Assay Buffer	Millipore	L-AB	Buffer
Low-protein binding storage plates	Thermo Scientific	CLS8161	Plates
Protease Inhibitor	Roche	11873580001	complete EDTA-free Protease Inhibitor Cocktail
Reader of multi-spot plates	Mesoscale	NA	Sector imager 6000
Assays	Mesoscale		Human 10-plex TH1/TH2 cytokine assay and 9-plex chemokine assay, and singleplex IL-17A, TGF-β1and TSLP. A description can be found online on www.mesoscale.com